ATP-sensitive potassium [KATP] channels in the heart muscle and coronary myocytes couple cellular metabolic status to membrane excitability, thereby contributing to the regulation of tissue responses to physiological and pathophysiological stimuli. In the heart muscle, opening of KATP channels participate in the stress response and protect against ischemic episodes. In the coronary vasculature, K(ATP/NDP) channels contribute to the regulation of basal flow as well as responses to metabolic impairment (hypoxic dilatation and ischemic reactive hyperemia). We found glycolytic enzymes to associate with KATP channel subunits, We hypothesize that glycolytic enzymes are integral components of the KATP channel macromolecular complex and that glycolytic enzymes regulate KATP channel activity under physiological and pathophysiological conditions, both in the cardiac myocyte as well as in the coronary smooth muscle and endothelium. In a first Specific Aim, we will investigate the hypothesis that enzymes of the glycolytic complex are associated with the KATP channel. Using co-immunoprecipitation assays we will investigate the specificity of interaction of glycolytic enzymes with individual KATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B). Co-immunoprecipitation assays of native proteins will be performed to investigate interactions under physiologically relevant conditions. Protein interactions will also be investigated using advanced proteomic approaches (ICAT &ITRAQ), which has the potential to uncover additional novel KATP channel interacting proteins. In a second Aim, we will examine the hypothesis that physical interaction of glycolytic enzymes with KATP channel subunits is required and that channel modulation occurs because of altered nucleotide levels in the microenvironment of the channel complex. This will be accomplished using mutant KATP channel subunits (lacking interaction with glycolytic enzymes or altered nucleotide sensitivity). In a final Aim, we will investigate the interaction of glycolysis and KATP channels in the context of ischemic protection in cardiac myocytes and the coronary vasculature. To this end, we will utilize our genetic mouse models that express dominant-negative K(ATP) channel subunits specifically in the cardiac myocyte, smooth muscle or endothelium. Our findings may have important implications for understanding the role of KATP channels in the heart and coronary vasculature under physiological and non-pathophysiological conditions. PUBLIC HEALTH RELEVANCE: In many cell types, there is an important link between intracellular energy metabolism and membrane excitability, which affects physiological process as diverse as insulin secretion, control of blood flow and protection of the heart against ischemia. We propose to examine one of the main effectors in this context, namely the ATP-sensitive K+ channel, and how the channel is specifically controlled by the initial step in glucose metabolism, namely glycolysis. Understanding this modality of channel regulation will further our knowledge of the known protective effects of the KATP channel in myocardial ischemia and cardiovascular disease.